We are using two DNA polymerases obtained by recombinant DNA technology, to examine the mechanisms and protein-DNA interactions that determine the fidelity of DNA synthesis. We have determined the fidelity of DNA synthesis catalyzed by the normal Klenow polymerase, by two mutant derivatives lacking proofreading exonuclease activity but having a normal protein structure, and by a protein that contains only the large polymerase domain. Measurements with the polymerases lacking an exonuclease show that the base-substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state kinetic measurements of selectivity at the insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proof-reading by the wild type enzyme improves the average base-substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for minus-one-base frameshift errors, with an error rate of <10-6. The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases. Upon removal of the small domain, the large polymerase domain was found to have altered fidelity for several classes of mutations. The fidelity results have also permitted the examination of a model to explain the production of minus-one base frameshift errors at non-reiterated base sequences. We have also established the fidelity of the thermostable Taq polymerase used in polymerase chain reactions (PCR), using various reaction condition, including changes in temperature, pH, relative and absolute dNTP concen- tration and MgCl2 concentration. These studies define high fidelity conditions that should be useful for genetic applications of DNA amplified by PCR.